CN112251528B

CN112251528B - Microsatellite DNA molecular marker for identifying ploidy of lily and application thereof

Info

Publication number: CN112251528B
Application number: CN202011119778.6A
Authority: CN
Inventors: 贾桂霞; 周艳萍; 李介文; 汪莲娟; 余鹏程; 何恒斌; 高雪
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2020-10-19
Filing date: 2020-10-19
Publication date: 2021-07-06
Anticipated expiration: 2040-10-19
Also published as: CN112251528A

Abstract

The invention relates to the technical field of DNA molecular markers, in particular to a microsatellite DNA molecular marker for identifying ploidy of lily and application thereof. The invention provides a microsatellite DNA molecular marker for identifying ploidy of lily, which comprises a combination of one or more of 9 microsatellite DNA molecular markers J1-044, J2-080, J3-091, J4-094, J5-096, J6-098, J7-117, J8-123 and J9-167. The invention also provides a primer for amplifying the 9 microsatellite DNA molecular markers, and establishes a method for carrying out lily ploidy identification by utilizing the microsatellite DNA molecular markers on the basis of the primer. The microsatellite DNA molecular marker and the identification method of the lily ploidy provided by the invention have the characteristics of good repeatability and high accuracy, and provide reliable and effective technical support for lily ploidy identification.

Description

Microsatellite DNA molecular marker for identifying ploidy of lily and application thereof

Technical Field

The invention relates to the technical field of DNA molecular markers, in particular to a microsatellite DNA molecular marker for identifying ploidy of lily and application thereof.

Background

Lily (Lilium spp.) is a generic name of all species of Lilium (Lilium) belonging to the family Liliaceae of the subclass monocotyledonous plant, the monocotyledonous subclass, is a world-famous bulbous flower, can be used for cut flowers, pot flowers and landscaping, and has a long medicinal and edible history.

The breeding of lily has a history of over one hundred years, and new varieties are continuously emerged almost every year. The parent material of lily breeding has a large amount of cultivated varieties as main parent materials except wild resources. The internationally popular Lily variety classification is mainly carried out by a classification system specified by Royal garden Society of great britain (Royal horticulture Society, RHS) and North American Lily Society (NALS), namely, classification is carried out according to genetic derivation relations, flower colors, flower patterns, posture characteristics and the like of different Lily cultivars and original relatives or hybrids thereof (longyayi, zhangjingguan Lily plant resource protection and utilization [ J ]. plant resource and environment, 1998(01): 40-44.). Currently, the main modern cultivar groups of lilies are as follows: asian lily hybrid lines (A), Oriental lily hybrid lines (O), Muscat lily hybrid lines (L), Trumpet and Aurelian hybrid lines (T), pure white lily hybrid lines (Candidum Hybrids), American lily hybrid lines (American Hybrids), Limonid leaf hybrid lines (Martagon Hybrids), and other hybrid lines [ e.g., Musca musk lily hybrid Lines (LA), east lily hybrid lines OT, east lily hybrid lines LO ], etc. ].

In modern commercial Lily varieties, the individual hybrid lines are more derived from intragroup crosses, Asian Lily hybrid lines (A) are from the rolling petiole group (Sinomartagon), Oriental Lily hybrid lines are from the petiole group (Archeliion), musk Lily hybrid lines are from the trumpet group (Leucoliron), except that the sub-hundred series partial varieties are triploid or tetraploid (Zhou S J. Aneuploid in Lily Breeding [ J ]. Acta Horticulturae.2014, (1027): 149-. In recent years, intergroup hybrid lines such as LA (asian lily × lilium musk), LO (lilium musk × lilium oriental), OT (lilium oriental × lilium trumpet) have been gaining increasing market favor, and many of these hybrids are obtained by distant hybridization, and F1 doubling or repeated hybridization after induction of 2n gametes, resulting in variety ploidy diversity. The existing research shows that the LA heterozygote is mainly triploid (Likehu, Zhouyixue, lingling, Zhangxian, Guo Fang, Zhou Shujun. Lily variety chromosome ploidy observation [ J ]. gardening academic newspaper.2011, 38(5): 970-; a few current reports of LO varieties are triploids (Likehu, Zhouyixue, Guilingling, Zhangling, Guoguang, Zhongjun. Lily variety for chromosome ploidy Observation [ J ]. Horticulture proceedings.2011, 38(5): 970-; OT Lily has both diploid, triploid and tetraploid (Emsweller S L, Uhring J. Lilium. X 'Black Beauty' and ampphipidloid [ J ]. Lily Yeast book.1966,29: 45-47.; Likehu, Zhonggui snow, Arcanling, Zhangian line, Guoguang, Zhongjun. Lily variety chromosome ploidy observation [ J. History, 2011,38(5): 970-. Therefore, ploidy identification of lily varieties has become a key problem to be solved first in lily breeding at present.

Disclosure of Invention

The invention aims to provide a microsatellite DNA (SSR) molecular marker for identifying ploidy of lily, and the invention also aims to provide application of the microsatellite DNA molecular marker.

In order to achieve the purpose, the invention carries out transcriptome sequencing on lily of a plurality of varieties, takes a sequence obtained by the transcriptome sequencing as a reference, and carries out SSR locus identification and positioning on Unigenes obtained by splicing and assembling; designing a primer for the screened Unigene sequence containing the SSR locus, and ensuring that the length of the flanking sequence of the SSR locus is more than or equal to 50 bp; the designed primers are further sequenced and compared, and 151 pairs of primers with high polymorphism are screened from more than twenty thousand pairs of primers. The method comprises the steps of further screening 151 pairs of primers obtained by screening 15 different varieties of lily, using a Touchdown PCR program for preliminary screening, obtaining 69 pairs of primers through total screening, synthesizing primers containing M13 joints, further re-screening, mainly using a two-step PCR program for re-screening, and finally selecting 43 pairs of core primers with higher polymorphism and more stable for subsequent tests. 43 pairs of core primers obtained by screening are utilized, genome DNA of 290 different lily varieties and wild species is used as a template to carry out SSR-PCR amplification, 9 microsatellite markers which have clear and stable bands and high polymorphism and can be used for lily ploidy identification are obtained by screening, the numbers of the microsatellite markers are J1-044, J2-080, J3-091, J4-094, J5-096, J6-098, J7-117, J8-123 and J9-167, and the primer sequences are respectively shown as SEQ ID NO. 1-18.

Based on the above findings, the present invention provides the following technical solutions:

the invention provides a microsatellite DNA molecular marker for identifying lily ploidy, which comprises a combination of one or more of microsatellite DNA molecular markers J1-044, J2-080, J3-091, J4-094, J5-096, J6-098, J7-117, J8-123 and J9-167; j1-044 is obtained by amplifying a primer shown by SEQ ID NO.1-2, J2-080 is obtained by amplifying a primer shown by SEQ ID NO.3-4, J3-091 is obtained by amplifying a primer shown by SEQ ID NO.5-6, J4-094 is obtained by amplifying a primer shown by SEQ ID NO.7-8, J5-096 is obtained by amplifying a primer shown by SEQ ID NO.9-10, J6-098 is obtained by amplifying a primer shown by SEQ ID NO.11-12, J7-117 is obtained by amplifying a primer shown by SEQ ID NO.13-14, J8-123 is obtained by amplifying a primer shown by SEQ ID NO.15-16, and J9-167 is obtained by amplifying a primer shown by SEQ ID NO. 17-18.

The primer sequences are specifically as follows:

SEQ ID NO.1：J1-044F:5'-CAGTATAATTAGTGACGTGCCTGG-3'

SEQ ID NO.2：J1-044R:5'-TCAATACTCTCACAATCCTCCAAA-3'

SEQ ID NO.3：J2-080F:5'-GGAGGGACTCTCGAGTATTTATCA-3'

SEQ ID NO.4：J2-080R:5'-GCTTCCTGTTTATCTCCACTGATT-3'

SEQ ID NO.5：J3-091F:5'-GTCATCACAATACCCTCTCTGGA-3'

SEQ ID NO.6：J3-091R:5'-CTCAGGTAACAGATCCTGCACAC-3'

SEQ ID NO.7：J4-094F:5'-GTCTCTCCTTCCCCATACCCTA-3'

SEQ ID NO.8：J4-094R:5'-AGTACAGCGAGGATCCGTACAT-3'

SEQ ID NO.9：J5-096F:5'-GTCTTTAAACCTCAGGCAACAAGT-3'

SEQ ID NO.10：J5-096R:5'-AGGACCTTGAACATATGTCTGTGA-3'

SEQ ID NO.11：J6-098F:5'-GTGTTGCTGCTCCATGTATTTAAC-3'

SEQ ID NO.12：J6-098R:5'-TACACTTCTCAATGTTCCCTTCAA-3'

SEQ ID NO.13：J7-117F:5'-TGTCTACAATCGAGGAAGTTGAAG-3'

SEQ ID NO.14：J7-117R:5'-GGTTACCTACATAGACCCTGTTGC-3'

SEQ ID NO.15：J8-123F:5'-TTTTTATCTCCTCGAGACTGATCC-3'

SEQ ID NO.16：J8-123R:5'-ATCTTTCTCTGCTGGTTCTCATTT-3'

SEQ ID NO.17：J9-167F：5'-ACCACATCAGATCCAAACAATG-3'

SEQ ID NO.18：J9-167R:5'-AGGTCATGCAGAGATCTTGTGTT-3'。

when lily ploidy identification is performed, any 1 of microsatellite DNA molecular markers J1-044, J2-080, J3-091, J4-094, J5-096, J6-098, J7-117, J8-123 and J9-167, or any combination of 2, 3,4, 5, 6, 7 and 8 or any combination of the above 9 molecular markers can be used.

When no lily variety is known, the ploidy determination is preferably performed by using the combination of the 9 microsatellite DNA molecular markers.

Preferably, the microsatellite DNA molecular markers for identifying lily ploidy described in the invention comprise a combination of J1-044, J2-080, J3-091, J4-094, J5-096, J6-098, J7-117, J8-123 and J9-167.

The information on the microsatellite DNA molecular markers described above is shown in Table 1.

TABLE 1 microsatellite DNA molecular marker information

Specifically, for Lilium regale, EST sequences of microsatellite DNA molecular markers J1-044, J2-080 and J6-098 are shown as SEQ ID NO.19-20 and 24; for lily varieties 'Sobang', EST sequences of microsatellite DNA molecular markers J3-091, J5-096, J7-117 and J8-123 are shown as SEQ ID NO.21, 23, 25 and 26; for lily variety 'white light No. two', the EST sequence of microsatellite DNA molecular marker J4-094 is shown as SEQ ID NO. 22; for the lily variety 'Tiny Todd', the EST sequence of the microsatellite DNA molecular marker J9-167 is shown as SEQ ID NO. 27.

The invention also provides a primer or a primer combination for amplifying the microsatellite DNA molecular marker, which is a combination of one or more pairs of primers selected from the primers shown in SEQ ID NO. 1-18.

For ease of detection, the primers described above may be fluorescently labeled. Alternative fluorescent labels include, but are not limited to, ROX, TRAMA, FAM, and the like.

The invention also provides a kit comprising the primer or the primer combination. The kit can be used for ploidy identification of lily.

Preferably, the kit comprises primers shown in SEQ ID NO. 1-18.

The kit described above may also include other components for PCR amplification including, but not limited to, PCR reaction buffer, dntps, DNA polymerase, negative controls, positive controls, and the like.

The invention provides application of the microsatellite DNA molecular marker or the primer combination or the kit in lily ploidy identification.

The invention provides application of the microsatellite DNA molecular marker or the primer combination or the kit in lily genetic breeding.

The invention provides application of the microsatellite DNA molecular marker or the primer combination or the kit in lily germplasm resource diversity analysis or germplasm resource improvement.

The invention provides a method for identifying lily ploidy, which is characterized in that genome DNA of lily is used as a template, the microsatellite DNA molecular marker is subjected to genotyping, and the lily ploidy is judged according to the genotyping result.

Specifically, the genome DNA of lily is used as a template, the primer or the primer combination is adopted for PCR amplification, and the lily is judged to be diploid or polyploid according to the type of a strip of a PCR amplification product.

Preferably, PCR amplification is performed with primers shown in SEQ ID NO. 1-18.

Preferably, the reaction procedure of the PCR amplification comprises: the first step is as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃, 30s at 72 ℃ and 15-20 cycles; 10min at 72 ℃; the second step is that: 5min at 95 ℃; 30s at 95 ℃, 30s at 52 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃.

The reaction system of the PCR amplification is as follows: first step (10 μ l): 2 XPower Taq PCR MasterMix 5. mu.l, forward primer 0.1. mu.l (10. mu.M), reverse primer 0.1. mu.l (10. mu.M), genomic DNA 1. mu.l, ddH₂O is complemented; second step (20 μ l): 2 XPower Taq PCR MasterMix 5.6. mu.l, reverse primer 0.15. mu.l, M13 fluorescent-labeled primer 0.15. mu.l, first-step reaction product 10. mu.l, ddH₂And (4) complementing O.

In the method, the sample to be detected is judged to be polyploid or diploid according to the number of bands of amplified fragments corresponding to each DNA polymorphic site of the sample to be detected, if the number of bands obtained by 1 DNA polymorphic site is more than two, the sample to be detected is judged to be polyploid, and if the number of bands obtained by 1 DNA polymorphic site is two, the sample to be detected is judged to be diploid.

The invention has the beneficial effects that: the invention provides 9 lily microsatellite loci and primers for amplifying the 9 microsatellite loci, and establishes a method for identifying lily ploidy by using lily microsatellite DNA molecular markers. The ploidy of the lily is identified by using the 9 microsatellite DNA molecular markers provided by the invention, the accuracy is high, the repeatability is good, and effective technical support is provided for lily polyploid breeding.

Drawings

FIGS. 1, 2 and 3 show the results of detection of J2-080 site amplification products of lily materials, sample Nos. A1, A25, A28, LA27, LA28, LA30, AT5, AT8, OT10, OT11, OT12, OT23, OT24, OT53, OT60, O1 and O3, in example 2 of the present invention.

FIGS. 4, 5 and 6 show the results of the detection of J5-096 site amplification products of lily materials with sample numbers A1, A25, A28, LA27, LA28, LA30, AT5, AT8, OT10, OT11, OT12, OT23, OT24, OT53, OT60, O1 and O3 in example 2 of the present invention.

FIGS. 7, 8 and 9 show the results of the detection of the J7-117 site amplification products from the lily materials with sample numbers A1, A25, A28, LA27, LA28, LA30, AT5, AT8, OT10, OT11, OT12, OT23, OT24, OT53, O1 and O3 in example 2 of the present invention.

In fig. 1, 2, 3,4, 5, 6, 7, 8 and 9, the abscissa represents the size of the amplification product and the ordinate represents the amplification intensity.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 identification of SSR sites in Lily transcriptome sequences and design of microsatellite molecular marker primers

The invention carries out transcriptome sequencing on lily of a plurality of varieties, takes a sequence obtained by the transcriptome sequencing as a reference, and carries out SSR locus identification and positioning on Unigenes obtained by splicing and assembling; designing primers for the Unigene sequence containing SSR sites obtained by screening, and gradually screening the primers to finally determine the microsatellite molecular marker which has less non-specific strip amplification, stable result and rich polymorphism and can be used for lily ploidy identification, wherein the specific method comprises the following steps:

1. taking a large number of sequences obtained by sequencing three groups of lily transcriptomes as reference, and identifying and positioning SSR sites of Unigenes obtained by splicing and assembling by utilizing MISA (http:// pgrc. ipkgatersleen. de/MISA) tool. The screening criteria were: the number of times of the single nucleotide repeat is 10 or more than 10, the number of times of the dinucleotide repeat is 5 or more than 5, the number of times of the trinucleotide repeat is 4 or more than 4, and the number of times of the four to six nucleotide repeat is 3 or more than 3. At the same time, incompletely repeated SSRs interrupted in the middle by a few bases (spacing less than 100 or equal to 100) were also screened.

2. And (3) designing a Primer for the screened Unigene sequence containing the SSR locus by using a Primer 3.0 Primer batch design program, and ensuring that the length of the flanking sequence of the SSR locus is more than or equal to 50 bp. The main parameters of primer design are as follows: the length of the primer sequence is 18-24 bp, the length of the PCR product is 100-300 bp, the GC content is 45-65%, and the Tm value is 55-75 ℃, so that hairpin structures, dimers, mismatching and primer dimers are avoided as much as possible.

3. Reference (Chen C, Bock client H, Beckman Tom G. sequence analysis methods EST-SSR primer performance and polymorphism [ J]2014,289: 1147-1156), the primer sequences of the three data sets designed were sequenced and compared in Microsoft Excel, and the same primer sequences from the three data sets, having the same repeat motif and the same amplicon, were first deleted. Secondly, comparing the repeat motifs, product length, three types that may occur are as follows: (1) if the repeated motifs are the same and the product lengths are different, the polymorphism is inferred to exist, and the polymorphism is selected; (2) the products have the same length and different repeated motifs, and are inferred to have polymorphism and are selected; (3) if the two are the same, the selection is not carried out. For example, a pair of identical primers, upstream and downstream, from the New Cannon and Minjiang data sets, respectively, the repeat motif of New Cannon is (CCCGGC)₄The repeat motif Minjiang 242, CCCGGC₃The product length is 236, and this type is the same as the above-mentioned repeat motif and the product length is different, and it is presumed that it has a polymorphism as an inclusion primer. By using the method, the primer screening range is greatly reduced, and 151 microsatellite markers are finally selected.

4. Further screening of lily microsatellite primers:

(1) the primer primary screening mainly uses a Touchdown PCR program, and the secondary screening mainly uses a two-step PCR program. The amplification system is shown in Table 2.

Touchdown PCR program: 5min at 95 ℃; 30s at 95 ℃, 30s at 65-55 ℃ (1 ℃ per cycle), 30s at 72 ℃ and 10 cycles; 30s at 95 ℃, 30s at 55 ℃, 30s at 72 ℃ and 20 cycles; 72 ℃ for 10min, and finally 4 ℃ for heat preservation.

Two-step PCR procedure:

the first step is as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃, 30s at 72 ℃ and 20 cycles; 72 ℃ for 10min, and finally 4 ℃ for heat preservation.

The second step is that: 5min at 95 ℃; 30s at 95 ℃, 30s at 52 ℃, 30s at 72 ℃ and 35 cycles; 72 ℃ for 10min, and finally 4 ℃ for heat preservation.

TABLE 2 PCR amplification reaction System formulation

(2) Two methods were used to detect the PCR amplification products: 8% non-denaturing polyacrylamide gel electrophoresis and fluorescence capillary electrophoresis, and respectively performing primary screening and secondary screening on the primers. Initially screening SSR-PCR amplification with 15 lily variety genome DNAs as templates, detecting the polymorphism of each primer of each PCR product on 8% polyacrylamide gel, and screening 69 microsatellite markers with less nonspecific strip amplification, stable result and rich polymorphism. And (3) further rescreening, wherein a two-step PCR program is mainly utilized for rescreening, and 43 pairs of core primers with higher polymorphism and more stability are finally selected for subsequent tests. 43 pairs of core primers obtained by screening are utilized, genome DNA of 290 different lily varieties and wild species is used as a template to carry out SSR-PCR amplification, a fluorescence capillary electrophoresis method is adopted to screen out 9 microsatellite DNA molecular markers with less nonspecific strip amplification, stable result and rich polymorphism, the serial numbers of the components are respectively J1-044, J2-080, J3-091, J4-094, J5-096, J6-098, J7-117, J8-123 and J9-167, the amplification primers are respectively shown in SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16 and SEQ ID NO.17-18 in sequence, and the 9 microsatellite DNA molecular markers are suitable for identifying ploidy of lily.

Example 2 identification of ploidy of different varieties of Lilium brownii by Using Lily microsatellite DNA molecular markers

The ploidy of different varieties of lily is identified by using the 9 microsatellite DNA molecular markers obtained by screening in the embodiment 1, and the specific method is as follows:

(1) extracting 116 genome DNAs of different lily varieties (novel plant genome DNA rapid extraction kit DN15, purchased from Beijing Ederly Biotech Co., Ltd.);

(2) PCR amplification was carried out using specific primers for J1-044, J2-080, J3-091, J4-094, J5-096, J6-098, J7-117, J8-123, and J9-167 sites, respectively, and using genomic DNAs of 116 lily varieties as templates, and the PCR amplification system is shown in Table 3:

TABLE 3 PCR amplification reaction System recipe

The two-step PCR procedure was as follows:

The sequences of the forward and reverse primers used were as follows:

SEQ ID NO.1：J1-044F:5'-CAGTATAATTAGTGACGTGCCTGG-3'

SEQ ID NO.2：J1-044R:5'-TCAATACTCTCACAATCCTCCAAA-3'

SEQ ID NO.3：J2-080F:5'-GGAGGGACTCTCGAGTATTTATCA-3'

SEQ ID NO.4：J2-080R:5'-GCTTCCTGTTTATCTCCACTGATT-3'

SEQ ID NO.5：J3-091F:5'-GTCATCACAATACCCTCTCTGGA-3'

SEQ ID NO.6：J3-091R:5'-CTCAGGTAACAGATCCTGCACAC-3'

SEQ ID NO.7：J4-094F:5'-GTCTCTCCTTCCCCATACCCTA-3'

SEQ ID NO.8：J4-094R:5'-AGTACAGCGAGGATCCGTACAT-3'

SEQ ID NO.9：J5-096F:5'-GTCTTTAAACCTCAGGCAACAAGT-3'

SEQ ID NO.10：J5-096R:5'-AGGACCTTGAACATATGTCTGTGA-3'

SEQ ID NO.11：J6-098F:5'-GTGTTGCTGCTCCATGTATTTAAC-3'

SEQ ID NO.12：J6-098R:5'-TACACTTCTCAATGTTCCCTTCAA-3'

SEQ ID NO.13：J7-117F:5'-TGTCTACAATCGAGGAAGTTGAAG-3'

SEQ ID NO.14：J7-117R:5'-GGTTACCTACATAGACCCTGTTGC-3'

SEQ ID NO.15：J8-123F:5'-TTTTTATCTCCTCGAGACTGATCC-3'

SEQ ID NO.16：J8-123R:5'-ATCTTTCTCTGCTGGTTCTCATTT-3'

SEQ ID NO.17：J9-167F：5'-ACCACATCAGATCCAAACAATG-3'

SEQ ID NO.18：J9-167R:5'-AGGTCATGCAGAGATCTTGTGTT-3'；

(3) performing STR genotyping on the PCR product by using an ABI3730XL genetic analyzer (Applied biosystems), and judging specific numerical values of alleles by using GeneMarker2.0 software;

(4) the characteristics of the lily DNA polymorphic sites are described by using Popgen 1.32 to calculate the values of the allelic gene factors and the polymorphic information content, and the results are shown in Table 4:

TABLE 49 characteristics of the Lilium microsatellite polymorphic sites

(5) And (4) judging a result: for 9 microsatellite DNA molecular markers, if more than two alleles are detected, the individual is judged to be possible to be polyploid, otherwise, the individual is diploid.

The names of the 116 different lily varieties and the information of hybrid lines belonging to the materials are shown in table 5, and the results of amplifying the 116 lily varieties by using 9 microsatellite DNA molecular markers are shown in table 6, table 7, table 8 and table 9, wherein the results of detecting J2-080 site amplification products of lily materials with sample numbers of A1, A25, A28, LA27, LA28, LA30, AT5, AT8, OT10, OT11, OT12, OT23, OT24, OT53, OT60, O1 and O3 are shown in fig. 1, fig. 2 and fig. 3; the detection results of J5-096 site amplification products of lily materials with sample numbers A1, A25, A28, LA27, LA28, LA30, AT5, AT8, OT10, OT11, OT12, OT23, OT24, OT53, OT60, O1 and O3 are shown in FIG. 4, FIG. 5 and FIG. 6; the results of detection of J7-117 site amplification products of lily materials with sample numbers A1, A25, A28, LA27, LA28, LA30, AT5, AT8, OT10, OT11, OT12, OT23, OT24, OT53, O1 and O3 are shown in FIG. 7, FIG. 8 and FIG. 9.

The results showed that 78 of the 116 parts of lily material were determined to be polyploid by detecting 3 or 4 alleles at least 1 time, and 38 parts of lily material were determined to be diploid by detecting only 1 or 2 alleles.

TABLE 5116 names of different lily varieties

Note: a represents Asian lily hybrid lines (Asiatic hybirds), AP represents potted Asian lily hybrid lines, AT represents Tier series and Pearl series Asian lily hybrid lines (Asiatic-Tiger Hybrids, Asiatic-Pearl Hybrids); o represents an Oriental lily hybrid line (Oriental hybrids); LO represents a filial generation of the hybrid of the musk lily hybrid and the oriental lily hybrid; LA represents a progeny of a cross of the musk lily hybrid line with an asian lily hybrid line; m represents lilium metschnikoides hybrid (Martagon hybrids); f represents a new lilium formolongi hybrid line; SP is a domesticated variety of the original strain; w represents the original species (species).

TABLE 69 amplification of primers in 116 germplasm materials (statistics of 3 and more than 3 amplification products only)

TABLE 79 amplification of primers in 116 germplasm materials (statistics of 3 and more than 3 amplification products only)

TABLE 89 amplification of primers in 116 germplasm materials (statistics of 3 and more than 3 amplification products only)

Table 99 amplification of primers in 116 germplasm materials (statistics of 3 and more than 3 amplification products only)

*: the ploidy results predicted by flow cytometry differed from the results described in the literature

(6) Analysis of accuracy of ploidy identification: to further determine ploidy, 89 parts of the material were selected for flow cytometry and conventional root tip tableting. 45 species and varieties were verified by flow cytometry, 35 species and varieties were verified by root tip pelleting, and the remaining 27 varieties were ploidy determined by literature review. The results showed that of 116 material, 41 material was diploid (35.65% by weight), 49 material was triploid (42.61% by weight), 25 material was tetraploid (21.74% by weight), and 1 material was further verified for ploidy. The 9 microsatellite DNA molecular markers correctly predict the diploid or polyploid attributes of 109 materials in 116 lily varieties to be tested, and the accuracy reaches 93.97%. Therefore, the microsatellite DNA molecular marker can be used for rapidly identifying the ploidy of the lily, has good repeatability and high accuracy, and is a reliable and effective DNA molecular marker.

The references cited in tables 6-9 are as follows:

1. likehu, Zhouyuixue, Guilingling, Zhangxian, Guo fang, Zhou Zjun, Lily variety ploidy observation [ J ] Horticulture proceedings 2011,38(5): 970-.

2、Van Tuyl J M,Arens P.Lilium:Breeding history of the modern cultivar assortment[J].Acta horticulturae.2011,900.

3. Shouxing clock, Qi-allotetraploid lily (Lilium) fertility analysis [ D ]. Master academic paper of Jiangxi agriculture university.2019.

4. Hopcalite, white and splendid, sinus-bright, lang-lixin, poroid, still grand and loyal.karyotype analysis of 8 LA lily varieties [ J ]. proceedings of shanxi university of agriculture (natural science edition). 2018,38(3):57-62.

5. Summer crystal, different ploidy lily (Lilium spp.) hybrid progeny embryo rescue and chromosome analysis [ D ]. master paper of southwest university 2010.

6、Cao Q Z,Lian Y Q,Wang L J,Zhang Q,Zhao Y Q,Jia G X,He H B.Physical mapping of 45S rDNA loci in Lilium OT hybrids and interspecific hybrids with Lilium regale[J].Scientia Horticulturae.2019,252:48-54.

7. Wei late, Wang Zhong Xuan, Jiagui Xia, OT series lily 3 varieties of karyotype analysis [ J ]. university of northeast forestry, proceedings of 2013,41(11):59-62.

8、Emsweller S L,Uhring J.Lilium×'Black Beauty'—diploid and amphidiploid[J].Lily Year Book.1966,29:45-47.

9、Van Tuyl J M,Arens P,Shahin A,Marasek-Ciolakowska A,Barba-Gonzalez R,Kim H T,Lim K B.Lilium[J].Ornamental Crops.2018,481-512.doi:10.1007/978-3-319-90698-0_20。

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtcatcacaa taccctctct gga 23

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctcaggtaac agatcctgca cac 23

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtctctcctt ccccataccc ta 22

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agtacagcga ggatccgtac at 22

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtctttaaac ctcaggcaac aagt 24

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aggaccttga acatatgtct gtga 24

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gtgttgctgc tccatgtatt taac 24

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tacacttctc aatgttccct tcaa 24

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgtctacaat cgaggaagtt gaag 24

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggttacctac atagaccctg ttgc 24

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tttttatctc ctcgagactg atcc 24

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atctttctct gctggttctc attt 24

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

accacatcag atccaaacaa tg 22

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aggtcatgca gagatcttgt gtt 23

<210> 19

<211> 477

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cttggtgaaa aaggaccgtg gccgtccaag cgaccctaaa gctaggccaa aagcatttgg 60

agaagtcagt ataattagtg acgtgcctgg cgtagaatta ctagaacatg atgatggtga 120

tgaagaagaa gatgatgatt ctacgcatct tagttctgat aatgagggtg atggttctga 180

tagtgctcac aatgatgtag tagaggaaga ggaagaggaa gaggaagagg aagaggaaga 240

ggaagatcta gaatccgatg atgagaaagt agatgcagat gatttggagg attgtgagag 300

tattgatgaa gataatgatc ttttagatgc tagtgattct ggggatgaga cgtctgatga 360

tgaaaatcag gttgaagacg ttgataatga tggatctgaa aaagtcaaag cactttgtag 420

tgagtcagac aatgacggcc atgatgatga aaaggaaata gtcaagtccg taacagg 477

<210> 20

<211> 641

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

agattacaag caccatgggt ttgggaggga ctctcgagta tttatcagat ctgtttggaa 60

gcagcggcca cacgcacaag aagaagaaga agcagttgca gactgtggag ctcaaggtga 120

ggatggactg cgatgggtgt gagctcaaag tcaagaaaac cctatcttcc atgtcaggag 180

tgaaatcagt ggagataaac aggaagcagc agaaggtaac tgtgacggga tatgtggagc 240

caaagaaggt gctgaagaag gctcagtcaa cagggaagaa ggctgagatc tggccttatg 300

ttccatacag cttggtgtct cagccctata ttgcaggaac ctacgacaaa aaagcccctc 360

ctgggtatgt gaggtatgtt gagcctgttg ctgtctcgag ctatgcaagc aaacaggagg 420

accagatcac caacatgttc agtgatgaca atccaaattc ctgctctatc atgtgagagg 480

aatggagtac atgagtctac cgtgtggaag gagtaggcag aactaatcat ggggcagtta 540

aatattgctt gtttgtaccc agggtttaga cggaaatgag tgggttggta tgtgacagct 600

acatgttagg taacgagttt tttataatta tctgttgtgt a 641

<210> 21

<211> 458

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gcagcttgcg agtcacgcaa cgagaccggt cccgctcgga gctcagcgcc gccgttgcgg 60

gaacccggcg ccgtcgtcat cacaataccc tctctggaga ccgagcattc ggcactgacg 120

ggcggtgcga aggttaacgc ggcggcggcg ccgaaggcgc tggtggacga gcaacggccg 180

gggaggccgg cggcggcggg ggatagtgtg gcggtcgatt tggggacgag tggcggatgc 240

gggggtctgg acggcgagac ggtgtgcagg atctgttacc tgagcccggg ccgcggggat 300

gcggcggtgg agatgatcga tctcggctgc cggtgcaagg atgaactcgg gaccgcgcac 360

cggcactgcg ccgaggcttg gttcaggatc aagggcaaca ggtgctgtga aatttgtggc 420

acgaacgcaa agaacataac cagtttggaa aatggctg 458

<210> 22

<211> 1814

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tctcccccca tttccgccgt ctctccttcc ccatacccta gctcctcgtc ctcgtcctcg 60

tcctcctcct cctcctcctc catgtccaac cacatgtacg gctacggaac gagctacggt 120

ggcggcgccg gaggctacgg ccagcgtgaa aacccctacc tctccgacgc cggcaggtac 180

ggtgtctccg gtcagctctc tgccgctgcc tccgctggca tgtacggatc ctcgctgtac 240

tcgagccagg ccgaacgata cggtctcccc agcagcgtcg gcgccggggg atacgtcggg 300

gcagcggcgc cgtccccgtt ctcgcagaac ccgtggtcgg cagcggctgc ggacccgacg 360

gctggcgcga agcgatctgc tgatgctctg taccatcaga atatcttggg aagcaatact 420

attggacaaa ctgaagctat attttcgaca aatcctttga tcaaacgccc tagatatgat 480

atagccagca atctgccgat atatccacag agaccgggag agaaggattg tcctcactat 540

atgttgacaa gaacctgtaa atttggagaa gcttgcaagt ttgaccatcc tgtttgggtt 600

ccagagggtg gggtcccaga ctggaaagag atccaaattg tcccgaacag cgaatctctt 660

cccgagagac caggggaacc tgattgtcct tactatatga agacacaaaa gtgcaagttc 720

ggattgcggt gcaagttcaa ccacccaaag gacaaactga atgtttcttc tggtggagtg 780

gtcacggaat ccattgacag agcagagtta cctgagagac catctgagcc aatttgttct 840

ttctatgcaa agacgggatt gtgcaagttt ggtgccacct gtaaatttca tcacccaaag 900

gacattcaaa tactatcagc tgggcaggat agtggtaatg ccggacaggg actaaacggt 960

agcgatgcag ctggtggaga cacaaaccca attaagacat ttgttccatt tactgcggcc 1020

ttgctgcata actcgaaggg gctacctata cgaccgggtg aaacagactg cccgttctac 1080

ctaaaaactg gcagttgcaa gtttggtgct acatgtcgat tcaaccaccc tgagagagca 1140

atcattccac cagttctagc gcctttaggg cattctgtcc tgtcttctaa tccagctaat 1200

tttcccattg gtgtctatgg tactgctaac attctcccaa atgcagccca agcattgctc 1260

ggtgtcaatt caaataccta tcctcaaagg ccagggcaaa cagaatgtga ttactatatg 1320

aagtccggac actgcagctt tggtgaacgg tgcaagtttc atcaccctat tgaccgcaca 1380

accccaacgg tgacaacaaa gcatggtccg gttccgaatg tcaagctaac ccttgcgggc 1440

ctcccaagaa gagagggttc agttgcatgt cccttctaca tgaaaactgg catttgcaag 1500

tatggtgcta cctgcaaata tgatcatcca ccaccaggtg aggctgtagc tttggcaaat 1560

gttcaaggag ccggaactac tgcaacagca ggagagcaga atgattctcc tgatgctcag 1620

gaacagtaga atgagtcagg cctgttccgt ttgtttcttt gcggcgcatt attagtttta 1680

ttgtatgtga gttaatataa tgcaactctg tttgttggga ttgatcaact atccttctgt 1740

taagtgtggg gagaaccatt gctgtctgaa tttgttagat cttggcattt taagatgaat 1800

ttattattct ggtt 1814

<210> 23

<211> 658

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gattgcagca gattggccac aatcaatgca aattgttcgc cttatagctc aatatctcat 60

gaataataaa caatacgtac gtaaggcgga ttacctggta tttcggacac taaatacaca 120

tggattcttg ggacaactgc aagaacagaa gctttgtgcc gttatccagc taccatcaca 180

aacactgctg ctctctgttt cagacaaggc aggacggttg atcggcatgc tctttcccgg 240

ggacatggtt gtctttaaac ctcaggcaac aagtcagcag caacaacaga tggtcttagg 300

ttcaggcatg aaccagcagc agcagcagca gcacatggga atgagccagc aaccacaaca 360

aatggttggg cctggtatga gccagcaaca aatgtcacag acatatgttc aaggtcctgg 420

gaggtctcag ttgatgtctc aggggaaaat gtcatcacac gggcctggtg gtaacatgtc 480

ttctggcgga tttttacctg atgggcatca ctagttgatc agagacttat tttgtattga 540

attattgggg ttttttctgt ctacttttgg tcctaacata actttggaat ttacaatatt 600

tcaaaatatt ccaaggtcgt tttcgacaac caaatctgtt atcgtattaa tttgaagc 658

<210> 24

<211> 1172

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tgaaaataga aagttttgga tatctcgacg tggtccaccg tgtggtgttg tcggcctgaa 60

aagcattggg ctgctcggta cctctctcag cacttccggt gttgctgctc catgtattta 120

acaccgatga gaaggggcaa acctacggcg atggcgatgg cgatggcgat gtccctctgt 180

ttctcttatt cttacgcccc acaggcacag ttgaagggaa cattgagaag tgtatcccat 240

cggtctctga gtactagtac agattttggt ggtctgcatt ttaagcatca gcttcggtgg 300

agttttgttg gtggctcaca acttgtgtgc ctgcctagag tcgctcgtac ggtctgctgc 360

cacagaaaag ggtccactct ttcagcttgt ttgtttcctg tagggctcac aagttctcaa 420

attgctaaca gtgcttttac gtggggaacg gttactgtcc ttccgttcta cacgctgatg 480

gttttggctc ctaacagtga actgactaaa agaagcatgg aaagcagtgt accatatgtt 540

ctgctcagtg tgctgtatgc ataccttcta tatctctcgt ggactcctga caccttgcgc 600

tctatgtttg caagcaaata ctggctgcca gagttacctg gcatagcaag aatgttctca 660

aatgaagtaa cagtggcgtc tgcgtggatt catttactgg ttgtcgatct tttcgctgca 720

agacaggtat ttcatgatgg tttaaagaag aagatagaga cccgacattc tgtttccttg 780

tgtctgctct tctgtccggt cggaatcctc gctcacgtca tcactaagct tctccccaag 840

acggagaacc catcgcattg aagaatgctt gtgttaggta atgggtgtgt atttagtgct 900

tggcttgttt aagctgtcta tgagaaacaa ggattctcag gagataaaat acttcattag 960

aagcccaggt tcttattagt ttatccatac tcaaatgtca tatgctagtt cttgattcga 1020

cggattacaa gtcaactaag ttccttcata tttggcttca aatgcttgtt tatgaaaagc 1080

cggtaaccat tcacaatgtg gataagaccc ataaagagac gagccggatg gatgatgaaa 1140

atgtgataaa attctcattt caaaaaaaaa aa 1172

<210> 25

<211> 318

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gtgacaatgt ctacaatcga ggaagttgaa gcagctgctc agcagttcaa tggttatgaa 60

cttgggggga gagcattgag ggtgaatgca ggaccacctc cgcctaaaga tgaagtccca 120

atgagaggat ttcgatcggg gcctggtggt ggcggcggcg gcggcggcgg tggtggttac 180

agttcgagca acagggtcta tgtaggtaac ctctcttggg gagttgacaa tgcatctctt 240

gagtccctgt tcaatgaaca aggaagggtc atggaagcca aggttgtgta cgacagggag 300

agtggccgat cgaggggc 318

<210> 26

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cttcttctcc tcgagcttgt tggggccgaa gatttggagc cggttggccc cctctgcctc 60

tgacaaaccc tccggactgc atttcagctg cacgaatact tcctccacag gaattcgttc 120

cagatcgacg gtctcgtttt ttatctcctc gagactgatc cccttatccg tcgccatcgc 180

cgccgccgat gatcagcaga tatctccgct gcggccgaag atcagcagag atcgccgccg 240

ccgccgctgc cgcggatcag cagagagggc agaactgtga ccgaacccta gacgaaatga 300

gaaccagcag agaaagatga g 321

<210> 27

<211> 1327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

caagctttca ccaaatatca ttcattggac tgaactaatt tttcagacat acgaagagac 60

agatcccaaa atccacagta tataacagct taacttttaa caatcaacac ggctaaataa 120

agcttaacag ccaacacgac caagaaccca cattttgatc catgattttt catcaaaaat 180

acttacaagc caaaagaatt gatgtatgca gatcagagca gaaactgtac acataaaaga 240

gaatgaatgc gccacaatat ctaatctaac ctttgctcgc caccgacttg cagacaggac 300

aagcattttt ctgaagaagc cattgcttga tacagtatgt atggtagcta tgcccacaat 360

aaagccttcc catctcatca ttcacttcat actcttcctg gcaaatgcag cattttcttt 420

ccatttcgga gaagcaaaat gaagggccag tgaaaaccgg gttcttcgat ttccgaaggc 480

agcacaaaat ctcttcttct cttaaaccag tactgacata cccaatttta tcgcctagct 540

caagcagttc ctcatatgac atgccatcca catcaagtcg caagtccctg tattggtcat 600

atgcgtcaat tgcgcccagc agaagtcgcg tttgaaacat cataatctct tcaaggccac 660

caggggaccg gcgatatccc cggatgtgcc ggagctgccc ggagggcacc acatcagatc 720

caaacaatgg cgtctcttga tcagatgacg catcaatgaa agcggagagc cgttcttgat 780

ttccgcctcc gccgccgccg ccgccgccgc gtcgtgcaac acaagatctc tgcatgacct 840

aaatcatcaa atcttgttca tatcacaata agagtgaatt acctacctga acccgatcgg 900

aatcagctct tccccctcgg acgacacagt cgacgggaac gtcggcggcg aaggcaatcc 960

cgggggcgca ccagacgtca gatgttgcgg cggcggcggg gcggcggaga tccttggcct 1020

tcttcttctg cttgactcga cggcggcgcg agcgcttgcc tggccagtcg gcggaggatc 1080

ggacggctgc ggcggcaggg gcgaagacct gggaggagga ggcggaggcg caaccgaggc 1140

cgcggagggc ggcgaagggc ttcttcttgg cggacggggg atcggggatg aaggggttgc 1200

ggtcggaggg ggtaaggcgg tttttgagga ggaggaggcg gcggattgtg ctggtggcgg 1260

cgtttggggc tgcctctgtc tccgccgcgg tcgccattga agtgaggggg atggggctct 1320

gttggtg 1327

Claims

1. Microsatellite DNA molecular markers for identifying ploidy of lily, characterized in that it comprises J1-044, J2-080, J3-091, J4-094, J5-096, J6-098, J7-117, J8-123 and J9-167,

j1-044 is obtained by amplifying a primer shown by SEQ ID NO.1-2, J2-080 is obtained by amplifying a primer shown by SEQ ID NO.3-4, J3-091 is obtained by amplifying a primer shown by SEQ ID NO.5-6, J4-094 is obtained by amplifying a primer shown by SEQ ID NO.7-8, J5-096 is obtained by amplifying a primer shown by SEQ ID NO.9-10, J6-098 is obtained by amplifying a primer shown by SEQ ID NO.11-12, J7-117 is obtained by amplifying a primer shown by SEQ ID NO.13-14, J8-123 is obtained by amplifying a primer shown by SEQ ID NO.15-16, and J9-167 is obtained by amplifying a primer shown by SEQ ID NO. 17-18.

2. A primer combination for amplifying the microsatellite DNA molecular marker of claim 1, which is a combination of primers shown in SEQ ID NO. 1-18.

3. A kit comprising the primer combination of claim 2.

4. Use of the microsatellite DNA molecular marker of claim 1 or the primer combination of claim 2 or the kit of claim 3 in lily ploidy identification.

5. Use of the microsatellite DNA molecular marker of claim 1 or the primer combination of claim 2 or the kit of claim 3 for lily genetic breeding.

6. Use of the microsatellite DNA molecular marker of claim 1 or the primer combination of claim 2 or the kit of claim 3 for lily germplasm resource diversity analysis or germplasm resource improvement.

7. A method for identifying lily ploidy, characterized in that, using lily genome DNA as a template, carrying out genotyping on the microsatellite DNA molecular marker of claim 1, and judging the lily ploidy according to the genotyping result.

8. The method according to claim 7, wherein the genomic DNA of lily is used as a template, the primer combination according to claim 2 is used for PCR amplification, and the lily is judged to be diploid or polyploid according to the type of the bands of the PCR amplification product.

9. The method of claim 8, wherein the reaction sequence for PCR amplification comprises: the first step is as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 60 ℃, 30s at 72 ℃ and 15-20 cycles; 10min at 72 ℃; the second step is that: 5min at 95 ℃; 30s at 95 ℃, 30s at 52 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃.